The in vitro cell culture system has been widely used as a primary screening tool for evaluating anti-HIV-1 activity of microbicides. However, there is clearly a need to develop a vaginal and cervical tissue based in vitro system to test the cytotoxicity and antiviral activity in the context of the complete tissue matrix. We have recently developed a cervical tissue-derived organ culture model which mimics in vivo conditions and has been used to test microbicides for their ability to block HIV-1 transmission. We also have preliminary data to show that this model can be used to monitor inflammatory cytokines in response to microbicides and sexually transmitted infection-related bacteria. Our hypothesis is that a cervical tissue-based organ culture is an ideal system to test microbicides for toxicity in genital tissue and for its ability to block transmission of HIV-1 with varying phenotypic properties across the cervical epithelium in the presence of common environmental factors that are present in vagina, such as semen, vaginal fluid, and STI-related microorganisms. Specific aims of the project are: 1) Evaluation of potential microbicides from Projects 1 (Cynanovirin-expressing lactobacillus) and 2 ( antimicrobial peptide retrocyclin). These microbicides will be tested for their ability to block HIV-1 transmission across the mucosa in a cervical tissue-based organ culture. In addition, the effect of confounding substances, such as semen and vaginal fluids from women will be tested on the anti-viral activity of these microbicides in organ culture, thus mimicing the in vivo condition; 2) Assessment of cervical tissue inflammation and their changes in response to microbicides from Project 1 and 2 using the organ culture model. The expression of proinflammatory cytokines, such as II-1beta, IL-6, IL-8 and TNF-alpha will be monitored by measuring their messages in the tissues by the real time PCR and secretion in the culture supernatant using the Luminex system; 3) Evaluation of anti-HIV activity of microbicides in the presence of other reproductive STI-related microorganisms, such as Neisseria gonorrhoeas. Cervical-tissue based organ culture model will be expanded to measure vaginal and cervical inflammation in response to HIV-1 and these reproductive STI-related microorganisms and lactobacilli, as a negative control. The proposed studies in this project will complement various other projects in this U19 grant application by providing a valuable in vitro cervical tissue-based assay which will bridge between the microbicide development (Projects 1 and 2), monkey model (Project 4), formulation (Core) and FDA approved additional antiviral testing core.